Determining a single analyte in a mixture of ˜100,000 other components is a formidable task. More than 60 years ago, analysts began to recognize that the structural selectivity of antibodies could be used to bind and purify antigens and haptens from biological extracts or blood on the basis of their chemical structure. This technique became so important that Rosalyn Yalow was awarded the Nobel Prize for radio-immunological assays (RIA) in 1960. Along with enzyme linked immunosorbent assays (ELISA), these two technologies provided the world with a simple method to measure antigens down to the pg/mL level.
A fundamental component of both RIA and ELISA is the use of immobilized antibodies to achieve antigen selection from samples in the first step of an assay. Since the inception of these approaches, analytical immunologists have understood that binding antibodies at a surface introduces significant kinetic limitations. For instance, antigens have to travel substantial distances in terms of molecular dimensions to reach surfaces, thereby adding to the amount of time needed for antigen binding to occur in a test tube or microtiter well. Moreover, all antibodies used in an assay are bunched together at the surface of an assay well or on a particle, while antigens are uniformly distributed throughout the solution. When an ELISA is carried out in a microtiter well, incubation times of a day or more are typically used to allow time for the antigens to diffuse to the walls of the well where the antibodies are bound. Efforts to minimize the diffusion problems in the case of RIA involved using large numbers of very small inorganic particles to which antibodies were immobilized. Over the course of the past several years, many types of mixing, flow, heating, and even sonication procedures have been used to minimize the diffusion problem noted above. Despite these efforts, diffusion problems still exist.
As analytical chemistry has evolved, it has been recognized that better and more complete answers can be obtained to various questions involving a sample if multiple analytes are determined simultaneously. This in turn has led to an increased interest in “analytical multiplexing”, where large numbers of analytes are analyzed in a sample during the course of a single analysis. This is often done through immunological arrays.
Interest in immunological arrays stems from the popularity and success of micro-electro-mechanical-systems (MEMS). While several types of antibody arrays have been used for large scale multiplexing, including high throughput and parallel processing techniques, other approaches have focused on large scale multiplexing with smaller numbers of samples, such as would be needed in a clinical laboratory that is concerned about minimizing the total analysis time and cost per assay. No matter what approach is utilized, immunological array systems still face challenges with respect to antibody immobilization and kinetics. There are also issues with respect to whether antibodies will retain full activity after immobilization, particularly if they are improperly oriented at the surface. Steric issues must also be considered, particularly in terms of the orientation of the antibody to the surface, as well as its packing density. Finally, reproducibility is also a factor, particularly as it is very difficult to reproduce immobilized antibodies from picoliter volumes of solution deposited on a surface. Evaporation, as well as a myriad of other phenomena, also diminishes reproducibility from that experienced at the titer plate level of immobilization.
Although the distance antigens must diffuse to reach surfaces is smaller in an immunological array than in a microtiter well, kinetic issues are still a serious limitation with immunological arrays. This is particularly true at low antigen concentrations. A substantial amount of time is required for an antigen to diffuse from all points in the solution to one of the array elements. Because molecular docking in antigen:antibody complex formations requires precise spatial orientation, antigens generally strike surfaces many times before establishing the correct capturing orientation. For instance, if an antigen is not captured after colliding with the surface on a 128 element array, it has a lot of space to navigate before striking the surface a second time.
As noted above, particle based assays began with the RIA and Yalow approaches. Currently the Yalow approach has evolved into two types of assay systems: 1) a particle approach used in flow cytometry assays (e.g., the Luminex system) where the fluorescence of individual particles is examined; and 2) an approach where antibodies are placed on a magnetic particle where the immune complex is formed, and the particle is then pulled out of solution and the antigens released for further measurements (e.g., the SISCAPA system of Leigh Anderson). Multiplexing requires the preparation of a different set of immunosorbent beads for each antigen being determined. This means that 20-50 different sets of antibody carrying beads would need to be added to the sample solution, thereby causing even larger kinetic limitations of the antigens in terms of finding the appropriate antibody particle. In addition, the solution becomes crowded with so many particles that the antigens must diffuse around particles not carrying their antibody. One proposed solution for dealing with these limitations is to immobilize multiple antibodies on a single particle. However, this solution does not completely address the issues of diffusion and stoichiometric control. Moreover, the dilution of antibody concentration on the particle surface means that the antigens can strike particle surfaces, while not contacting their antibody. In addition, the total surface area, and thus the total number of particles required, would still remain high.
With respect to the flow cytometry strategy (such as the Luminex system), immune complexes must be formed on the particle surfaces before they can be analyzed by flow cytometry. While this system is very similar to the above, each bead carries a single antibody targeting a single antigen. Again, there is the diffusion problem in antigen capture.
It is interesting that the function of mammalian immune systems is to deal with thousands of antigens, albeit not all of them simultaneously. As immunity to foreign substances develops in an individual mammal, antibodies to thousands of immunogens are produced. These antibodies are contained within the immunoglobulins circulating in blood where at any time, hundreds of antigens are being sequestered as antigen:antibody complexes are formed. Upon analyzing mammalian immune systems, it can be concluded that antibodies have evolved to function in solution as they form immune complexes. In addition to functioning in solution, they also sequester large numbers of antigens at the same time and have few of the limitations seen within immobilized antibody assay systems.
The above-noted observations of mammalian immune systems are extremely important, particularly as they clearly suggest that the formation of immune complexes in solution are naturally efficient, while the formation of complexes on immobilized surfaces are not. Moreover, it is clear that several immune complexes can be simultaneously formed in a solution (such as blood), which is a necessary factor to achieve when performing an analytical multiplexing process. Finally, most of the problems that commonly impact immunological assays (e.g., loss of activity during immobilization, proper orientation of antibodies, diffusion kinetics and having sufficient surface area) are not prevalent within these natural solution based systems.
Despite the above-described advantages of naturally formed immune complexes, such immunological assays still require the addition of antibodies to the samples, which can be concerning. For instance, adding large numbers of antibodies to a plasma sample may cause the protein concentration to increase to such a level that analyte diffusion is hampered. While this issue may seem concerning on its face, upon taking a further look, it can be concluded that this issue is likely inconsequential. More particularly, the average concentration of serum albumin in plasma is present in the range of about 50 to about 100 mg/mL, while immunoglobulins are present in an amount of approximately 4 mg/mL, and that of any particular antibody is probably in the range of from about 1 to about 100 ug/mL. If it is assumed that the concentration of an antibody needed to carry out an analytical measurement is 10 ug/mL and when 100 antibodies are to be added to a plasma sample, the total increase in protein concentration would be about 1 mg/mL. Similarly, if the concentration of protein in the plasma sample were 75 mg/mL, the increase in protein concentration would be about 1.3%. As such, it can be concluded that the addition of 100 antibodies to plasma to carry out a 100-fold multiplexed analysis would have almost no effect on protein concentration, solution viscosity, and ultimately analyte diffusion. Moreover, adding a thousand antibodies would only add 10 mg/mL of mass, or a 14% change in protein concentration; which again, would likely not be enough to impact the analysis.
Prior to 1960, immunological assays generally targeted individual antigens and were carried out in solution through a process called the ‘precipitin reaction’. Subsequent to immune complex formation, the polyclonal antibody mixture being used in the assay either formed a precipitate, or was induced to do so by the addition of a carbohydrate or an ethylene glycol polymer. Antigen concentration was determined by light scattering; however, the lack of sensitivity and linearity associated with this approach, as well as the fact that the precipitin assay only permitted one antigen to be assayed at a time, led to the demise of the method, and ultimately a transition to the far more sensitive RIA and ELISA methods. Despite the failures of the precipitin reaction method, it can still be reasoned that solution based immune complex formation approaches could be useful for immunological assays if selectivity and detection sensitivity were vastly improved.
Although the original precipitin and RIA approaches depended on a single method of selection (i.e. one antibody) in the execution of antigen measurements, it is accepted today that a sole antibody is not sufficiently selective to discriminate between an antigen and all the other chemical entities in a sample. Current immunological assays are built on multiple dimensions of selection and/or discrimination, and it is particularly ideal if each of these dimensions is of orthogonal selectivity.